Chronic Inflammation Alters Production and Release of Glutathione and Related Thiols in Human U373 Astroglial Cells

Neurons rely on glutathione (GSH) and its degradation product cysteinylglycine released by astrocytes to maintain their antioxidant defences. This is particularly important under conditions of inflammation and oxidative stress, as observed in many neurodegenerative diseases including Alzheimer’s disease (AD). The effects of inflammatory activation on intracellular GSH content and the extracellular thiol profile (including cysteinylglycine and homocysteine) of astrocytes were investigated. U373 astroglial cells exposed to IL-1β and TNF-α for up to 96 h showed a dose-dependent increase in IL-6 release, indicative of increasing pro-inflammatory cellular activation. With increasing concentrations of IL-1β and TNF-α (0.01–1 ng/ml), an increase in both intracellular and extracellular GSH levels was observed, followed by a return to control levels in response to higher concentrations of IL-1β and TNF-α. Extracellular levels of cysteinylglycine decreased in response to all concentrations of IL-1β and TNF-α. In contrast, levels of the neurotoxic thiol homocysteine increased in a dose-dependent manner to IL-1β and TNF-α-induced activation. Our results suggest that chronically activated astrocytes in the brain might fail to adequately maintain GSH substrate delivery to neurons, thus promoting neuronal vulnerability. They might also explain the elevated levels of homocysteine found in the brains and serum of patients with AD.     

Chronic sequelae complicate convalescence from both dengue and acute viral respiratory illness

Long Covid has raised awareness of the potentially disabling chronic sequelae that afflicts patients after acute viral infection. Similar syndromes of post-infectious sequelae have also been observed after other viral infections such as dengue, but their true prevalence and functional impact remain poorly defined. We prospectively enrolled 209 patients with acute dengue (n = 48; one with severe dengue) and other acute viral respiratory infections (ARI) (n = 161), and followed them up for chronic sequelae up to one year post-enrolment, prior to the onset of the Covid-19 pandemic. Baseline demographics and co-morbidities were balanced between both groups except for gender, with more males in the dengue cohort (63% vs 29%, p<0.001). Except for the first visit, data on symptoms were collected remotely using a purpose-built mobile phone application. Mental health outcomes were evaluated using the validated SF-12v2 Health Survey. Almost all patients (95.8% of dengue and 94.4% of ARI patients) experienced at least one symptom of fatigue, somnolence, headache, concentration impairment or memory impairment within the first week of enrolment. Amongst patients with at least 3-months of follow-up, 18.0% in the dengue cohort and 14.6% in the ARI cohort experienced persistent symptoms. The median month-3 SF-12v2 Mental Component Summary Score was lower in patients who remained symptomatic at 3 months and beyond, compared to those whose symptoms fully resolved (47.7 vs. 56.0, p<0.001), indicating that patients who self-reported persistence of symptoms also experienced functionally worse mental health. No statistically significant difference in age, gender distribution or hospitalisation status was observed between those with and without chronic sequelae. Our findings reveal an under-appreciated burden of post-infection chronic sequelae in dengue and ARI patients. They call for studies to define the pathophysiology of this condition, and determine the efficacy of both vaccines as well as antiviral drugs in preventing such sequelae.     

Isolation of two isoforms of a novel 15-kDa protein from rabbit polymorphonuclear leukocytes that modulate the antibacterial actions of other leukocyte proteins

We have recently reported the use of the highly selective and reversible binding of the potent bactericidal/permeability-increasing protein (BPI) to target Gram-negative bacteria (Escherichia coli) for its isolation from crude extracts of human polymorphonuclear leukocytes (PMN). We now report the use of the same procedure for the purification from rabbit PMN of BPI and also of a novel 15-kDa species that consists of two nearly identical isoforms. These 15-kDa proteins have no demonstrable antibacterial activities by themselves. However, one isoform (p15A) potentiates strongly and the other (p15B) weakly the early antibacterial effects of both rabbit and human BPI. Both isoforms inhibit the late lethal action of BPI. Whereas the potentiating effect is specific for BPI the inhibitory effect is seen also with another antibacterial protein of PMN granules, azurocidin. Thus, we have identified in rabbit PMN a previously unrecognized 15-kDa protein species that may modulate during phagocytosis the antimicrobial effects of BPI (and other granule proteins).     

Examination of protein sequence homologies: II. SixEscherichia coli L7/L12-type ribosomal “A” protein sequnces from eukaryotes and metabacteria,contrasted with those from prokaryotes

Summary Four complete and three partial sequences ofE. coli L7/L12-type ribosomal A proteins obtained from four eukaryotes (Saccharomyces cerevisiae, Artemia salina, rat liver, and wheat germ), two metabacteria (Halobacterium cutirubrum andMethanobacterium thermoautotrophicum), and the prokaryoteEscherichia coli have been compared using a computer program that searches for homologous tertiary structures. Comparison matrices show that eukaryotic sequences sequentially match each other if deletions and/or insertions of certain residues (gaps) are assumed at specific sites corresponding to residues 36, 51, 72, and 94 ofS. cerevisiae protein YL44c. This is similar to what was previously found in prokaryotes. Metabacteria, which exhibit eukaryote-type sequences, must have separated from the eukaryotes in ancient times, because an additional deletion site is found in their sequences and their sequences have low correlation coefficients with those of all the other eukaryotes. When the eukaryote-type A proteins (110–111 residues) are compared withE. coli L7/L12 (120 residues) four groups of well-matching segments are found. It was deduced that the eukaryote-type A proteins had regenerated from the prokaryote types by a transposition and several deletions, resulting in the eukaryote-type lengths. The correspondence between the eukaryotic and prokaryotic proteins, as well as that among eukaryotic proteins themselves, is discussed in terms of protein evolution.In addition, ribosomal protein YL35 fromS. cerevisiae has been compared with RL37 from rat liver, with results indicating five well-matching parts separated by four gaps, one of which consists of 20 residues. These results contrasts with those previously reported by Lin et al. No prokaryotic counterparts to these ribosomal proteins have yet been identified.     

Relation between sequence similarity and structural similarity in proteins. Role of important properties of amino acids

In a previous paper we obtained ten (orthogonal) factors, linear combinations of which can express the properties of the 20 naturally occurring amino acids. In this paper, we assume that the most important properties (linear combinations of these ten factors) that determine the three-dimensional structure of a protein are conserved properties, i.e., are those that have been conserved during evolution. Two definitions of a conserved property are presented: (1) a conserved property for an average protein is defined as that linear combination of the ten factors that optimally expresses the similarity of one amino acid to another (hence, little change during evolution), as given by the relatedness odds matrix of Dayhoff et al.; (2) a conserved property for each position in the amino acid sequence (locus) of a specific family of homologous proteins (the cytochromec family or the globin family) is defined as that linear combination of the ten factors that is common among a set of amino acids at a given locus when the sequences are properly aligned. When the specificity at each locus is averaged over all loci, the same features are observed for three expressions of these two definitions, namely the conserved property for an average protein, the average conserved property for the cytochromec family, and the average conserved property for the globin family; we find that bulk and hydrophobicity (information about packing and long-range interactions) are more important than other properties, such as the preference for adopting a specific backbone structure (information about short-range interactions). We also demonstrate that the sequence profile of a conserved property, defined for each locus of a protein family (definition 2), corresponds uniquely to the three-dimensional structure, while the conserved property for an average protein (definition 1) is not useful for the prediction of protein structure. The amino acid sequences of numerous proteins are searched to find those that are similar, in terms of the conserved properties (definition 2), to sequences of the same size from one of the homologous families (cytochromec and globin, respectively) for whose loci the conserved properties were defined. Many similar sequences are found, the number of similarities decreasing with increasing size of the segment. However, the segments must be rather long (15 residues) before the comparisons become meaningful. As an example, one sufficiently large sequence (20 residues) from a protein of known structure (apo-liver alcohol dehydrogenase that is not a member of either family) is found to be similar in the conserved properties to a particular sequence of a member of the family of human hemoglobin chains, and the two sequences have similar structures. This means that, since conserved properties are expected to be structure determinants, we can use the conserved properties to predict an initial protein structure for subsequent energy minimization for a protein for which the conserved properties are similar to those of a family of proteins with a sufficiently large number of homologous amino acid sequences; such a large number of homologous sequences is required to define a conserved property for each locus of the homologous protein family.     

Identification of Novel Cytotoxic Peptide KENPVLSLVNGMF from Marine Sponge <Emphasis Type="Italic">Xestospongia testudinaria</Emphasis>, with Characterization of Stability in Human Serum

Resistance and side effects are common problems for anticancer drugs used in chemotherapy. Thus, continued research to discover novel and specific anticancer drugs is obligatory. Marine sponges hold great promise as a source of potent cytotoxic peptides with future applications in cancer treatments. This study aimed to purify and identify cytotoxic peptides from the protein hydrolysates of the giant barrel sponge Xestospongia testudinaria, guided by a cytotoxicity assay based on the human cervical cancer cell line (HeLa). Comparison among trypsin, chymotrypsin, papain and alcalase hydrolysates of X. testudinaria revealed papain hydrolysate (PH) to be the most active. PH was purified consecutively by membrane ultrafiltration, gel filtration chromatography, and reversed-phase high performance liquid chromatography (RP-HPLC). Following liquid chromatography-tandem mass spectrometric analysis, two peptides were identified from the most cytotoxic RP-HPLC fraction: KENPVLSLVNGMF and LLATIPKVGVFSILV. Between the two, only the synthetic peptide KENPVLSLVNGMF showed cytotoxicity toward HeLa cells in a dose-dependent manner. KENPVLSLVNGMF (EC₅₀ 0.67 mM) was 3.8-fold more cytotoxic compared with anticancer drug 5-fluorouracil (EC₅₀ 2.56 mM). Furthermore, KENPVLSLVNGMF show only marginal 5% cytotoxicity to Hek293, a non-cancerous, human embryonic kidney cell line, when tested at 0.67 mM. The half-life of the peptide was 3.2?±?0.5 h in human serum in vitro, as revealed by RP-HPLC analyses. These results suggest that KENPVLSLVNGMF identified from X. testudinaria papain hydrolysate has potential applications as peptide lead in future development of potent and specific anticancer drugs.     

Oxygen-deficiency and allelochemicals affect Sphagnum spore persistence in peatlands

Plant and Soil - To test the effects of characteristic ecological gradients in peatlands including oxygen-deficiency and allelopathy on Sphagnum spore persistence. We determined the initial...